The function of the stacking tryptophan, W290, a second coordination sphere residue in galactose oxidase has been investigated via steady-state kinetics measurements, absorption, CD and EPR spectroscopy, and x -ray crystallography of the W290F, W290G, and W290H variants. Enzymatic turnover is significantly lower in the W290 variants. The K m for D-galactose for W290H is similar to wild type, whereas the Km is greatly elevated in W290G and W290F, suggesting a role for W290 in substrate binding/positioning via the -NH group of the indole ring. Hydrogen bonding between W290 and azide in the wild type-azide crystal structure are consistent with this function. W290 modulates the properties and reactivity of the redox-active tyrosine radical; the Y272 tyrosyl radical in both the W290G and W290H variants have elevated redox potentials and are highly unstable compared to the radical in W290F, which has similar properties to the wild type tyrosyl radical. W290 restricts the accessibility of the Y272 radical site to solvent. Crystal structures show that Y272 is significantly more solvent exposed in W290G variant but that W290F limits solvent access comparable to the wild-type indole side chain. Spectroscopic studies indicate that the Cu(II) ground states in the semi-reduced W290 variants are very similar to that of the wild-type protein. In addition, the electronic structures of W290X-azide complexes the variants are also closely similar to the wild type electronic structure. Azide binding and azide-mediated proton uptake by Y495 are perturbed in the variants, indicating that tryptophan also modulates the function of the catalytic base (Y495) in the wild-type enzyme. Thus, W290 plays multiple critical roles in enzyme catalysis, affecting substrate binding, the tyrosyl radical redox potential and stability, and the axial tyrosine function.Over the past twenty years, there has been a growing appreciation for the catalytic utility of protein-derived free radical cofactors in enzymes (1-3). Free radical chemistry is harnessed to catalyze bond activation and molecular rearrangements in a wide variety of enzymes including ribonucleotide reductase (4-7), DNA photolyase (8), cytochrome c peroxidase (9), pyruvateformate lyase (10), lysine-2,3-aminomutase (11), prostaglandin H synthase (12), glyoxal oxidase (13), and galactose oxidase (14).*Authors to whom correspondence should be addressed. Email: dmdooley@montana.edu, Tel: 406-994-4373, FAX: 406 -994-7989; Email: m.j.mcpherson@leeds.ac.uk, Tel: +44 113 233-2595, FAX: +44 113 233-3167. 1 Data deposition: The atomic coordinates and structure factors for W290G, W290F and W290H have been deposited in the Protein Data Bank, www.rcsb.org. † This work was supported by a grant from the National Institutes of Health (GM27659 DMD) and from the Biotechnology and Biological Sciences Research Council (MJM). NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2008 September 9. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptIt...
Galactose oxidase (GO; EC 1.1.3.9) catalyses the oxidation of a wide range of primary alcohols including mono-, oligo- and polysaccharides. High-resolution structures have been determined for GO, but no structural information is available for the enzyme with bound substrate or inhibitor. Previously, computer-aided docking experiments have been used to develop a plausible model for interactions between GO and the D-galactose substrate. Residues implicated in such interactions include Arg330, Gln406, Phe464, Phe194 and Trp290. In the present study we describe an improved expression system for recombinant GO in the methylotrophic yeast Pichia pastoris. We use this system to express variant proteins mutated at Arg330 and Phe464 to explore the substrate binding model. We also demonstrate that the Arg330 variants display greater fructose oxidase activity than does wild-type GO.
Ethyl 2-acrylamido-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate as well as its corresponding bis-derivatives, 5-10, with aliphatic linkers were synthesized, fully characterized, and tested as novel anticancer agents. The targeted compounds, 5-10, were obtained by the Knoevenagel condensation reactions of bis-o-or-p-aldehyde with a molar ratio of ethyl 2-(2-cyanoacetamido)-4,5,6,7tetrahydrobenzo[b]thiophene-3-carboxylate of 2 in the presence of piperidine in excellent yields (93-98%). The in vitro anticancer activities of the prepared compounds were evaluated against HepG2, MCF-7, HCT-116, and BJ1 cells. Compounds 7 and 9 emerged as the most promising compounds, with IC 50 values of 13.5 and 32.2 µg/ml, respectively, against HepG2 cells, compared with the reference drug doxorubicin (IC 50 : 21.6 µg/ml). Real-time reverse-transcription polymerase chain reaction was used to measure the changes in expression levels of the COL10A1 and COL11A1, ESR1, and ERBB2, or AXIN1 and CDKN2A genes within the treated cells, as genetic markers for colon, breast, or liver cancers, respectively. Treatment of the colon cancer cells with compounds 5, 9, and 10, or breast and liver cancers cells with compounds 7, 8, 9, and 10 downregulated the expression of the investigated tumor markers. The DNA damage values (depending on comet and DNA fragmentation assays) increased significantly upon treatment of colon cancer cells with compounds 5, 9, and 10, and breast and liver cells with compounds 8, 9, and 10. The structure-activity relationship suggested that the increase of the chain of the alkyl linker increases the anticancer activity and the compounds with bis-cyanoacrylamide moieties are more active than those with one cyanoacrylamide moiety.
Two new flavonoid compounds were isolated from Astragalus bombycinus Boiss. and identified as quercetin-3,7-di-O-β-glucopyranoside 4'-O-α-rhamnopyranoside and 5,2',4'-trihydroxy-flavone-8-C-α-arabinopyranoside-7-O-β-glucopyranoside. In addition, apigenin, apigenin-7-O-β-glucopyranoside, apigenin 7-O-gentobioside, luteolin, luteolin-7-O-β-glucopyranoside, quercetin-3,7-di-O-β-glucopyranoside, quercetin-3-O-β-glucopyranoside-7-O-α-rhamnopyranoside and daidzein were also isolated and identified. The structure elucidation of the isolated compounds was performed by chromatographic, chemical and spectroscopic methods. Antioxidant and cytotoxic activities were also determined for the four consecutive extracts of the plant.
Objective: Mulberry is a nontoxic commonly eaten plant, belongs to the Morus and used in folk medicine in the remedy of dysentery, antiphlogistic, diuretic, expectorant, and antidiabetic. The purpose of this study is to evaluate the antiproliferative and radical scavenging activity of the total alcoholic and successive fractions thereof of Morus alba and Morus rubra fruits. In addition, the chemical composition of the bioactive fractions of each species was investigated.Methods: The antiproliferative potential of 8 extracts on 4 human cancer cell lines, hepatocellular carcinoma (HepG2), Caucasian breast adenocarcinoma (MCF7), prostate (PC3), and colon carcinoma (HCT116) in addition to one normal cell line namely human normal immortalized skin fibroblast cells (BJ1) were carried out. Cell viability was determined using MTT assay. The potency was compared with the reference drug doxorubicin. These extracts were also assayed for 1,1-diphenyl-2-hydrazyl free radical scavenging activities. After saponification of the n-hexane fraction, unsaponifiable matter and fatty acid methyl esters were analyzed by gas liquid chromatography (GLC). The chemical composition of the bioactive fractions was investigated using gas chromatography/mass spectrometry (GC/MS) analysis.Results: All the extracts showed significant free radical scavenging activity dose-dependently. The n-hexane and dichloromethane (DCM) fractions of M. rubra exhibited potent cytotoxic activity on almost cancer cell lines. In the same pattern, ethyl acetate (EtOAc) of M. rubra has moderate cytotoxic activity against all cell lines except HepG2. DCM fraction of M. alba possessed both radical scavenging and high potential antiproliferated activities against HCT116 and MCF7 with inhibitory concentration of 43.9 and 32.3 μg/ml, respectively, while it showed no cytotoxic effect on BJ1. GLC analysis showed the major hydrocarbons in M. alba and M. rubra were heptacosane and docosane, respectively. Sterols were similar in both species but with different ratios and cholesterol was the major one. Palmitic and margaric were the major saturated fatty acid while arachidonic was the major unsaturated fatty acid in both species. GC/MS analysis showed the main compound in DCM fraction of each Morus species was palmitic acid. Furthermore, 1,11-bis-(methoxycarbonyl-ethenyl)-10,2-dihydroxy-cycloeicosane and linolelaidic acid, methyl ester were the main compounds in the EtOAc fraction of each Morus species. Whereas, the main compounds in alcoholic extract of M. alba and M. rubra were methyl-14-methyl-pentadecanoate and 1,2-O-isopropylyidene-4-nonene-1,2,3-triol, respectively.Conclusions: The results observed remarkable biological activity of the successive fractions of M. rubra more than those of M. alba and confirmed its importance as a natural bioactive source. Morus species are good candidates to be promising as possible sources for future antitumor and antioxidants in food and pharmaceutical formulations. The strong activity partly explains the potential effects of Morus species for the treatment of cancer and degenerative diseases caused by free radicals.
Microalgae represent a rich source that satisfies the growing need for novel ingredients of nutriceuticals, pharmaceuticals, and food supplements. Haematococcus pluvialis and Dunaliella salina microalgae are isolated from the Egyptian hydro-flora and are reported for their potent antioxidant activities. The cytotoxic activity of different fractions of both microalgae was investigated on 4 cell lines HePG2, MCF7, HCT116, and A549. The carotenoid rich fraction of H. pluvialis showed potent cytotoxic activity against colon cancer cell line and moderate activity against both liver and breast cancer cell lines. On the other hand, the carotenoid rich fraction of D. salina showed mild cytotoxic activity on breast and liver cancer cell lines. The carotenoid rich fraction of H. pluvialis was analysed using LC-DAD/ESI-MS and the major carotenoids were identified either free as well as bounded to fatty acids.
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